Infectious bursal disease (IBD) or Gumboro disease is a highly contagious viral disease of young chickens which is characterized by the destruction of lymphoid follicles in the bursa of Fabricius. In a fully susceptible chicken flock of 3-6 weeks of age the clinical disease causes severe immunosuppression, and is responsible for losses due to impaired growth, decreased feed efficiency, and death. Susceptible chickens less than 3 weeks old do not exhibit outward clinical signs of the disease but have a marked infection characterized by gross lesions of the bursa.
The virus associated with the symptoms of the disease was called infectious bursal disease virus (IBDV). IBDV is a pathogen of major economic importance to the nation and world's poultry industries. It causes severe immunodeficiency in young chickens by destruction of precursors of antibody-producing B cells in the bursa of Fabricius. Immunosuppression causes increased susceptibility to other diseases, and interferes with the effective vaccination against Newcastle-disease, Marek's disease and infectious bronchitis disease viruses.
There are two known serotypes of IBDV. Serotype I viruses are pathogenic to chickens whereas serotype II viruses infect chickens and turkeys. The infection of turkeys is presently of unknown clinical significance.
Up until recently, the principal methods of controlling IBD in young chickens were by vaccination with an avirulent strain of IBDV or by transferring high levels of maternal antibody induced by the administration of live and killed IBD vaccines to breeder hens (Wyeth, P. J. and Cullen, G. A., Vet. Rec. 10-4, 188-193, (1979)).
In recent years field outbreaks of IBD, particularly in the eastern United States, have shown infection of poultry with variant viruses which are not completely neutralized by antibodies against standard serotype I IBDV (Rosenberger, J. K. et al, Proc. of the 20th National Meeting on Poultry Health and Condemnations, (1985); Snyder, D. B. et al, Proc. 23rd National Meeting on Poultry Health and Condemnations, Ocean City, Md. (1988)).
IBDV belongs to a group of viruses called Birnaviridae which includes other bisegmented RNA viruses such as infectious pancreatic necrosis virus (fish), tellina virus and oyster virus (bivalve molluscs) and drosophila X virus (fruit fly). These viruses all contain high molecular weight (MW) double stranded RNA genomes.
The capsid of the IBDV virion consists of at least four structural proteins. As many as nine structural proteins have been reported but there is evidence that some of these may have a precursor-product relationship. The designation and molecular weights of the four viral proteins (VP) are as shown in Table 1 below.
TABLE 1 ______________________________________ Viral Proteins of IBDV Viral Protein Molecular Weight ______________________________________ VP1 90 kDa VP2 41 kDa VP3 32 kDa VP4 28 kDa ______________________________________
An additional protein, VPX, of 47 kDa was determined to be a precursor of the VP2 protein.
The nucleotide sequences of IBDV serotype I (ST-C, standard challenge virus and attenuated virus BB) and serotype II obtained from turkeys (OH, Ohio strain) have been compared and have provided preliminary information thereof (Jackwood, D. J. et al, 69th Annual Meeting of the Conference of Research Workers in Animal Disease, Abs. no. 346, Chicago, Ill. (1988)).
Two segments of double stranded RNA were identified in the genome of IBDV. One contains 3400 base pairs and has a molecular weight of 2.06.times.10.sup.6, and the other contains 2900 base pairs and has a molecular weight of 1.76.times.10.sup.6. In vitro translation of the denatured genomic RNA of the virus has shown that the larger RNA segment encodes three structural proteins, i.e., VP2, VP3 and VP4, and the smaller RNA segment encodes only one protein, i.e., VP1.
Both genomic segments of an Australian strain of IBDV, that is different from the U.S. strains, were recently cloned and sequenced (Hudson, P. J. et al, Nucleic Acids Res. 14, 5001-5012, (1986); Morgan, M. M. et al, Virology 163, 240-243, (1988)). The complete nucleotide sequence of the larger segment has shown that these proteins are encoded in the order VP2, VP4 and VP3, and that they are contained in one open reading frame. In addition, further nucleotide sequence data confirmed that the smaller RNA segment encodes only the VP1 protein (Morgan, M. M. et al, Virology 163, 240-243, (1988)). This protein is a minor component of the virion and it is presumed to be the viral RNA polymerase. In IBDV, the VP1 protein binds tightly to both ends of the two genomic segments, and it effectively circularizes the molecule.
It has been recently demonstrated that the VP2 protein is the major host protective immunogen of IBDV, and that it contains the antigenic region responsible for the induction of neutralizing antibodies. The region containing the neutralization site has been shown to be highly conformation-dependent. The VP3 protein has been considered to be a group-specific antigen because it is recognized by monoclonal antibodies directed against it from strains of both serotype I and II viruses. The VP4 protein appears to be a virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins. However, the precise manner in which the proteolytic break up takes place is not yet clear.
The occurrence of antigenic variations among IBDV isolates has been repeatedly reported. The use of monoclonal antibodies (MCA) B29, R63, B69, 179, BK9 and 57 raised against different strains of IBDV led to the recognition of the occurrence of three distinct antigenic types of IBDV in the field in the U.S. These data are shown in Table 2 below.
TABLE 2 __________________________________________________________________________ AC-ELISA characterization of banked field isolates, laboratory/reference and vaccine strains of IBDV. Capture MCA IBDV No. Virus Source B29 R63.sup.a B69.sup.a 179.sup.a BK9 57.sup.a Tested Type __________________________________________________________________________ Banked Isolates: Pure Classic + + + + - - 60 Classic Pure DEL + + - + + - 76 Delaware Pure GLS + - - + - + 70 Pure GLS Laboratory virus: IM + + + + - - 1 Classic STC + + + + - - 1 Classic Edgar + + + + - - 1 Classic 2512 + + + + - - 1 Classic LUK + + + + - - 1 Classic F52/70 + + + + - - 1 Classic MD + + + + - - 1 Classic A/DEL + + - + + - 1 Delaware D/DEL + + - + + - 1 Delaware E/DEL + + - + + - 1 Delaware G/DEL + + - + + - 1 Delaware Vaccines: D78 + + + + - - 1 Classic Univax + + + + - - I Classic Bursine + + + + - - 1 Classic Bio-Burs + + + + - - 1 Classic Bio-Burs I + + + + - - 1 Classic IBD Blend + + + + - - 1 Classic Bursa-vac + + + + - - 1 Classic VI-Bur-G + + + + - - 1 Classic S706 + + + + - - 1 Classic __________________________________________________________________________ .sup.a MCA neutralizes.
Two of the MCAs discussed above, B69 and 57, made specifically against the Classic D78 and GLS strains of IBDV have been found by virus neutralization tests to neutralize only the parent virus. The third MCA, R63, also made against the IBDV Classic strain was shown to neutralize all serotype I IBDVs except the GLS variant virus. Two other MCAs, 179 and BK44, have been shown to be potent neutralizers of all serotype I IBDVs studied so far.
All serotype I IBDVs bind to MCA B29 in an antigen-capture enzyme-linked immunosorbens assay (AC-ELISA). However, the B29 MCA is not a neutralizing MCA. On the other hand, the B69 and R63 MCAs are both neutralizing MCAs. Predictions on new variants can be made on the basis of their reactivities with the B69 MCA. A virus that does not bind to this MCA in an AC-ELISA is very likely antigenically different from the standard type ("classic"), and would be termed as a variant virus. Neither the Delaware type E (E/DEL) nor the GLS variants of IBDV react with the B69 MCA. In addition, the E/DEL variant can be distinguished from the GLS variant virus on the basis of its reactivity with the R63 MCA. The GLS variant virus does not bind to the R63 MCA in AC-ELISA assay as is shown in Table 2 above.
The new GLS variant was recently discovered on the basis of antigen-capture ELISA tests (Snyder, D. B. et al, Proc. 23rd Nat. Meeting Poultry Health and Condem., Ocean City, Md. (1988)). This strain of IBDV is presently replacing the Delaware variant and has already become the most predominant IBDV type occurring in the Delmarva Peninsula. Data on IBDV types obtained with the monoclonal antibodies (MCAs) R63, B29, above are shown in Table 3 below.
TABLE 3 ______________________________________ Geographic Distribution of IBDV Types as Determined With an MCA R63, B69 and B29 Based AC-ELISA. % IBDV Type Total # of State Classic Delaware GLS Isolates ______________________________________ Delmarva 8 42 50 319 AL 15 67 18 52 NC 19 52 29 67 MS 20 70 10 10 GA 22 52 26 27 AR 36 45 19 53 TN 50 50 0 2 MO 50 50 0 2 IN 57 43 0 7 FL 73 20 7 15 CA 84 8 8 25 OK 87 0 13 8 TX 100 0 0 10 OR 100 0 0 7 MN 100 0 0 7 WA 100 0 0 3 VA 0 100 0 8
0 100 0 5 Total #: 627 ______________________________________
There are currently 9 "live" attenuated avirulent vaccines available in the market. All the vaccine strains react with the B29, B69 and R63 in MCAs AC-ELISA tests. These viruses, therefore, are classified as the "Classic" type, as shown in Table 2 above. The brand name of these vaccines and their sources are given in Table 4 below.
TABLE 4 ______________________________________ Vaccines for IBDV Vaccine Company ______________________________________ Clone-vac D78 Intervet America Univax American Sci. Lab. Bursine Salisbury Bio-Burs KeeVet Bio-Burs I KeeVet IBD Blend Ceva Bursa-vac Sterwin VI-Bur-G Vineland S706 Select ______________________________________
The above vaccine strains are not virulent like the variant viruses and they may be given "live". Thus, they do not have to be inactivated or "killed" in order to be used as vaccines. However, these vaccines are not fully effective in protecting against infection with variant viruses. A limited number of chickens immunized with the above vaccine strains are actually protected against challenge with Delaware (about 60%) and GLS (about 30%) variant viruses.
In addition, the immunization with the "Classic" strains of IBDV (see, Table 4) that is routinely conducted nowadays renders the immunized birds partially protected only against the Delaware (DEL) and the GLS variant viruses.
A "killed" IBDV vaccine is also available from Intervet Co. in Millsboro, Del. This vaccine is called "Breeder-vac" and contains standard ("classic"), Delaware and GLS variant virus types. The use of the above "live" and "killed" vaccines has the following disadvantages, among others.
The viruses have to be propagated in tissue culture, which is time-consuming and expensive.
In "killed" vaccines, the viruses have to be inactivated prior to use, which requires an additional expensive step.
If the "killed" vaccines are not properly inactivated, a risk of an outbreak of the disease exists and does not provide broad protection to birds against the virus variants and the ensuing disease.
Thus, there is a palpable need for an improved vaccine which is effective in the treatment of IBD caused by various pathogenic IBDV strains.